Today, a gene introduction technique has made remarkable progress and become indispensable for production of a genetically recombined protein obtained by introducing a foreign gene into animal cells, production of genetically recombined organisms and a gene therapy.
The most important matter for a gene introduction technique is to efficiently introduce a gene of interest into cells and to express it stably. Normally, for obtaining animal cells or animal individuals expressing a gene of interest (hereinafter referred to as “recombinant”), a cassette for expressing said gene (a cassette consisting of a promoter, said gene and poly(A) addition signal sequence, hereinafter referred to as “expression cassette”) and a cassette for expressing a drug resistant gene as a marker gene for the gene introduction are introduced into ontogenetic cells such as EG cells or ES cells and those recombinants stably expressing the introduced gene are selected. An instrument used for introducing the expression cassette of said gene and the cassette of the drug resistant gene into cells is called a vector. In order to efficiently obtain a recombinant stably expressing said gene, how efficiently said gene may be introduced into chromosomal DNAs of a host, i.e. cells or animal individual to which gene introduction is aimed, would become the crux and in this regard a kind of a vector to be used is the most important. What kind of a vector is used would greatly vary insertion efficiency into chromosomal DNAs of a host as well as efficiency of subsequent recombinant production.
Today, a vector utilized for introducing a gene of interest into animal cells or animal individual may largely be classified into a viral vector where a viral genome within viral particles is used and a non-viral vector. Characteristic features of both vectors are outlined hereinbelow in view of insertion into chromosomal DNAs.
A viral vector, as its name indicates, is one wherein an expression unit of a gene of interest is inserted into a viral genome to prepare a viral particle bearing said gene within its genome and said viral particle is infected to animal cells or a fertilized egg or occasionally directly to animal individual for introduction of said gene. This type of vector is further classified into a vector introducible into chromosomal DNAs of a host and a vector not introduced into chromosomal DNAs of a host but present as an episome. Examples of the former include oncoretrovirus, lentivirus and adeno-associated virus vectors. Examples of the latter include adenovirus and herpesvirus vectors.
These viral vectors are capable of introducing a gene of interest into cells with high efficiency since they utilize the ability to infect cells originally possessed by the virus. These viral vectors also have limitation in cellular species for introduction since they retain cellular species specificity originally possessed by the virus. Furthermore, when oncoretrovirus or lentivirus vector introducible into chromosomal DNAs of a host is used, there is a concern in safety such as contamination of an expression product of a gene of interest with viruses due to generation of replicable viral particles or production of innate retrovirus due to insertion of a reverse transcriptase into a genome.
On the other hand, a plasmid vector is primarily used as a non-viral vector. A plasmid vector is one where a plasmid, which was found as an extranuclear circular gene that is replicated and retained outside the E. coli chromosome, is used as a vector. A plasmid vector, even with insertion of a gene of interest, may easily be multiplied within E. coli and thus has commonly been used as a gene introduction vector for animal cells. However, a plasmid vector, which is a DNA per se, is difficult to be introduced into cells without physical treatment such as microinjection or electroporation. At present, other than the physical technique, the method for efficiently introducing a plasmid vector into cells includes calcium phosphate coprecipitation and complex formation with DEAE-dextran or cationic lipids. With these devises, efficiency of gene introduction into cells has gradually been improved but is still much inferior to the above method using a viral vector. Furthermore, the most important is that, when a gene of interest is introduced via a plasmid vector, a probability of insertion of said gene into chromosomal DNAs of a host is extremely low such that the vector transferred from cytoplasm into nucleus may accidentally be inserted while chromosomal replication. A plasmid vector, though with the defects described above, is superior to a viral vector from the aspect of safety and up till the present a recombinant obtained with this vector alone has been used for production of a recombinant protein.
In recent years, for improving one of the greatest drawbacks of a plasmid vector, i.e. poor insertion efficiency into chromosomal DNAs of a host, a vector utilizing a transposon has been developed which has an insertion mechanism into chromosomal DNAs of a host. A transposon, referring to a gene which transposes on a chromosome and has firstly been reported by Barbara McClintock, is found to be present on a chromosome of various organisms (e.g. Non-patent reference 1).
A transposon may largely be classified into two groups (cf. e.g. Non-patent reference 2). One is a retro transposon classified as class I and the other is a DNA transposon classified as class II. A class I retro transposon, present as a DNA on a chromosome, is once transcribed into an RNA which is then transformed into a complementary DNA (cDNA) by the function of a reverse transcriptase coded therein and the cDNA is re-inserted into a chromosome. Thus, this type of transposon tends to continually multiply its copies insofar as it has the activity. A retro transposon is further divided into two large groups based on the presence or absence of a reverse transcriptase. A retro transposon with no reverse transcriptase encoded therein is a non-autonomous transposon that is not capable of transposing by itself but transposes by borrowing an exogenous reverse transcriptase. The group with a reverse transcriptase encoded therein is further divided into two large groups based on the presence or absence of Long Terminal Repeat (LTR). A so-called retrovirus, which has an LTR sequence at the end of its genome and encodes a reverse transcriptase, is thought to be a kind of a retro transposon.
On the other hand, a class II DNA transposon is cleaved from the insertion site on a chromosome via the function of an enzyme catalyzing transposition, called transposase, encoded by itself and is re-inserted into different site. From such a mode of transposition, this type of transposon is also called “cut-and-paste” transposon. This type of transposon characteristically has a Terminal Inverted Repeat (TIR) of several to as long as several hundreds of bases at both ends of a transposon gene as well as a gene encoding transposase flanked by TIR sequences. A transposase as expressed recognizes and binds with the terminal TIR sequences and undertakes reactions of cleavage from chromosomal DNAs and insertion at a different site of the transposon to thereby allow for its transposition on a chromosome. A majority of transposons with the transposition activity has only been reported in bacteria, plants and insects with some exceptions. However, in 1997, Ivics et al. isolated a transposon from a salmon belonging to Tc1/mariner superfamily, repaired a gene encoding a transposase inactivated through accumulation of genetic mutations and eventually succeeded in regeneration of a transposase with the “cut-and-paste” activity, which is named “Sleeping Beauty” (cf. e.g. Non-patent reference 3). It was revealed that the “Sleeping Beauty” has the transposition activity not only in cells derived from fish but also in cells derived from mammals and its introduction rate into chromosomal DNAs reached a level of 80-folds higher than that of usual transfection (cf. e.g. Non-patent reference 4).
Table 1 shows active transposons belonging to Tc1/mariner superfamily reported up till the present. Among the active transposons shown therein, the “Sleeping Beauty” was revealed to have the highest transposition activity (cf. e.g. Non-patent reference 5) and, with the benefit of its property that no host-derived factor is necessary for expression of the transposition activity, is going to be used as a non-viral, efficient vector for gene introduction into animal cells or animal individual.
TABLE 1Major transposons belonging to Tc1/mariner superfamilyTIRSuperfamilyfamilysubfamilylengthOrganismTc1/marinerTc1Tc154CaenorhabditiselegansTc3462CaenorhabditiselegansSleeping225Atlantic salmonBeautymarinerMinos255Drosophila hydeiMos128Drosophila mauritianaHimar131Haematobia irritans
A transposon vector system developed by Ivics et al. is such that a plasmid, wherein an expression unit of a gene of interest is inserted into a transposon vector having at both ends TIR sequences derived from white cloud mountain minnow (Tanichthys albonubes), and a plasmid, wherein an expression unit of a transposase (“Sleeping Beauty”) necessary for transposition into chromosomal DNAs is inserted into a transposon vector, are simultaneously introduced into cells. With this method, those clones alone having a gene of interest in chromosomal DNAs of cells are selected so as to avoid, as characteristic feature of transposon, transposition after insertion into chromosomal DNAs. Besides, a transposon vector used in this system has relatively long TIR sequences, characteristic of Tc3 among Tc1/mariner superfamily, in which two transposase-binding sequences called “Direct Repeat (DR)” are present (FIG. 1).
As outlined above, a transposon vector, typically “Sleeping Beauty”, exhibits high insertion efficiency into chromosomal DNAs as well as a broad host spectrum in spite of a non-viral vector and thus is expected to be increasingly used as a vector for gene introduction in future.
While such a development of a vector for gene introduction has much progress, a great progress is also seen in a technique for inserting a gene at a specific site on DNAs or for providing deletion or replacement of a specific gene. A typical example of such techniques is to use a recombination mechanism called “Cre-Lox” and “Flp-FRT” recombination systems.
Cre-Lox recombination system is an application of a recombination mechanism found in bacteriophage P1 consisting of two elements, i.e. LoxP sequences consisting of 34 bases where recombination occurs and Cre, an enzyme (recombinase) undertaking a recombination reaction. In a recombination reaction with wild-type LoxP, in the presence of Cre, there may occur both a reaction wherein a DNA sequence flanked by LoxP sequences is deprived and a reaction wherein a circular DNA having LoxP sequences is inserted into LoxP sequences present on a different DNA. However, with wild-type LoxP, the reaction of deprivation of a DNA sequence flanked by LoxP sequences preferentially occurs as compared to the insertion reaction into LoxP sequences and thus it will be hard to expect the latter reaction to occur (FIG. 2). Today, in order to solve this problem, mutated LoxP sequences have been prepared and applied for insertion reaction or replacement reaction not expected with wild-type LoxP. Table 2 shows major mutated Lox sequences being used and their mutated sites. Cre-Lox system with these mutated Lox sequences has allowed for efficient DNA insertion at Lox sequences or replacement of a DNA sequence between Lox sequences (FIG. 3),
TABLE 2Major mutated Lox sequencesSequence (only sequence ofa single-strand is indicated)Cre-bindingCre-bindingType ofNamesiteSpacersitemutationLoxPATAACTTCGTATAGCATACATTATACGAAGTTATWild type Lox71TACCGTTCGTATAGCATACATTATACGAAGTTATLE mutant Lox66ATAACTTCGTATAGCATACATTATACGAACGGTARE mutant LoxATAACTTCGTATAGGATACTTTATACGAAGTTATSpacer2272mutant LoxATAACTTCGTATAGTATACATTATACGAAGTTATSpacer511mutant*: The underlined sequences indicate mutations from those of wild type.
Flp-FRT recombination system is an application of a recombination mechanism found in yeast (Saccharomyces cerevisiae) which consists of, like Cre-Lox system, two elements, i.e. FRT sequences consisting of 48 bases where recombination occurs and Flp, a recombinase undertaking a recombination reaction. With this recombination system, it is also possible to remove a DNA sequence flanked by FRT sequences through deprivation reaction or to insert a circular DNA having FRT sequences into FRT sequences.
Thus, once a specific sequence could be inserted into chromosomal DNAs, it is now possible to induce insertion, deprivation or replacement reaction of a gene on a specific region, i.e. on a sequence where a recombination reaction occurs, by utilizing the recombination systems as described above.    Non-patent reference 1: Richardson R D et al., Stem Cells, 20, 105-118, 2002    Non-patent reference 2: Finnegan, Curr. Opin. Genet. Dev., 2, 861-867, 1992    Non-patent reference 3: Ivics Z et al., Cell, 91, 501-510, 1997    Non-patent reference 4: Yant S R et al., Nat. Genet., 25, 35-41, 2000    Non-patent reference 5: Sylvia E J et al., Proc. Natl. Acad. Sci. USA, 98, 6759-6764, 2001